Search

Antibodies are key reagents used in vision research, indeed across biomedical research, but they often do not reveal the whole story about a sample. It is important for researchers to be aware of aspects of antibodies that may affect or limit data interpretation. Federal agencies now require funded grants to demonstrate how they will authenticate reagents used. There is also a push for recombinant antibodies, enabled by phage display technology awarded the 2018 Nobel Prize in Chemistry, which allow for thorough validation and a fixed DNA sequence. Here, we discuss how issues surrounding antibodies are pertinent to detecting myocilin, a protein found in trabecular meshwork and associated with a portion of hereditary glaucoma. Confirmation of myocilin expression in tissues and cell culture has been adopted as validation standard in trabecular meshwork research; thus, a discussion of antibody characteristics and fidelity is critical. Further, based on our basic structural understanding of myocilin architecture and its biophysical aggregation properties, we provide a wish list for the characteristics of next-generation antibody reagents for vision researchers. In the long term, well-characterized antibodies targeting myocilin will enable new insights into its function and involvement in glaucoma pathogenesis.

Purpose To elucidate functions of wild-type myocilin, a secreted glycoprotein associated with glaucoma. Methods Lysates of mouse eyes were used for immunoprecipitation with affinity-purified antibodies against mouse myocilin. Shotgun proteomic analysis was used for the identification of proteins interacting with myocilin. Colocalization of myocilin and tissue inhibitor of metalloproteinases 3 (TIMP3) in different eye structures was investigated by a multiplex fluorescent in situ hybridization and immunofluorescent labeling with subsequent confocal microscopy. Matrix metalloproteinase 2 (MMP2) activity assay was used to test effects of myocilin on TIMP3 inhibitory action. Results TIMP3 was identified by a shotgun proteomic analysis as a protein that was coimmunoprecipitated with myocilin from eye lysates of wild-type and transgenic mice expressing elevated levels of mouse myocilin but not from lysates of transgenic mice expressing mutated mouse myocilin. Interaction of myocilin and TIMP3 was confirmed by coimmunoprecipitation of myocilin and TIMP3 from HEK293 cells transiently transfected with cDNAs encoding these proteins. The olfactomedin domain of myocilin is essential for interaction with TIMP3. In the eye, the main sites of myocilin and TIMP3 colocalization are the trabecular meshwork, sclera, and choroid. Using purified proteins, it has been shown that myocilin markedly enhanced the inhibitory activity of TIMP3 toward MMP2. Conclusions Myocilin may serve as a modulator of TIMP3 activity via interactions with the myocilin olfactomedin domain. Our data imply that in the case of MYOCILIN null or some glaucoma-causing mutations, inhibitory activity of TIMP3 toward MMP2 might be reduced, mimicking deleterious mutations in the TIMP3 gene.

Purpose Primary open-angle glaucoma (POAG) is particularly common in older individuals and associated with pathologic degeneration of the trabecular meshwork (TM). We have shown previously that transplantation of induced pluripotent stem cell (iPSC) derived TM cells restores aqueous humor dynamics in young transgenic mice expressing a pathogenic form of human myocilin (Tg-MYOCY437H). This study was designed to determine if this approach is feasible in older mice with more pronounced TM dysfunction. Methods Mouse iPSC were differentiated toward a TM cell phenotype (iPSC-TM) and injected into the anterior chamber of 6-month-old Tg-MYOCY437H or control mice. IOP and aqueous humor outflow facility were recorded for up to 3 months. Transmission electron microscopy, Western blot, and immunohistochemistry were performed to analyze TM morphology, quantify endoplasmic reticulum (ER) stress, and assess TM cellularity. Results A 12 weeks after transplantation, IOP in iPSC-TM recipients was statistically lower and outflow facility was significantly improved compared to untreated controls. The number of endogenous TM cells increased significantly in iPSC-TM recipients along with the appearance of TM cells immmunopositive for a marker of cellular division. Morphologically, transplantation of iPSC-TM preserves ER structure 12 weeks after transplantation. However, myocilin and calnexin expression levels remain elevated in transplanted eyes of these 9-month-old Tg-MYOCY437H mice, indicating that ER stress persists within the TM. Conclusions Transplantation of iPSC-TM can restore IOP and outflow facility in aged Tg-MYOCY437H mice. This type of stem cell-based therapy is a promising possibility for restoration of IOP control in some glaucoma patients.

Purpose Primary open angle glaucoma-associated mutations in myocilin (MYOC) cause protein "nonsecretion," rendering secreted MYOC difficult to detect using conventional techniques. This study focused on developing and using an assay that can quickly and easily detect mutant MYOC secretion. Methods We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T and NTM-5 cells. Secreted and intracellular levels of MYOC eGLuc2 variants were evaluated by Western blotting and compared to untagged and FLAG-tagged MYOC constructs. Secreted and soluble intracellular MYOC eGLuc2 were measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from transfected cells to test the applicability of the assay for monitoring other MYOC variants. Results Myocilin eGLuc2 behaved similarly to untagged and FLAG-tagged MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 minutes after media change. Gaussia luciferase fused variants followed anticipated trends; nonpathogenic variants (D208E, G244V) were secreted at wild-type (WT) levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperature. Interestingly, however, this increase in secretion was independent of newly synthesized protein. Conclusions Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. This newly developed MYOC reporter system can be used to study engineered MYOC variants and potentially to identify modulators of MYOC secretion and function.

Homeostasis of aqueous humor (AH) drainage through the trabecular meshwork (TM) is essential for maintenance of normal intraocular pressure.1 Importantly, blockage or increased resistance to AH outflow through the TM leads to elevated IOP, which is a major risk factor for primary open-angle glaucoma,2,3 a leading cause of blindness in the United States.4 The conventional AH outflow pathway of human eyes through which most AH is drained consists of the TM, juxtacanalicular (JCT) tissue, and Schlemm's canal (SC).5 The cells derived from these different tissues of the AH outflow pathway exhibit endothelial and mesenchymal characteristics.5,6 Various physiological and etiological mechanisms have been reported to account for homeostatic and deregulated AH outflow, respectively, through the conventional or trabecular pathway.2,3,5,7–10 Importantly, abundant experimental evidence also points to a role for the cellular properties of TM and SC, including contractile characteristics, actin cytoskeletal organization, cell adhesive interactions, mechanical and mechanosensing properties, and extracellular matrix (ECM) organization and production, in regulating AH outflow through the TM.2 To explore additional intracellular molecular mechanisms regulating the TM cell contractile, cell adhesive, and biomechanical properties for a thorough understanding of both homeostatic and deregulated AH outflow and IOP, we hypothesized that Hic-5, a multidomain adaptor protein, may play a crucial role in regulation of TM cell integrin–matrix interactions, actin cytoskeletal organization, and glucocorticoid-induced gene expression, eventually influencing AH outflow through the TM. Hic-5 is an inducible focal adhesion (FA) protein that belongs to the family of paxillin proteins expressed abundantly in smooth muscle cells of various tissues.11–14 Hic-5 was originally isolated as a hydrogen peroxide (H2O2)- and TGF-β1-inducible (TGFB1I1) gene that encodes a 55-kDa protein.11,12,15 Interestingly, in separate studies, this protein has also been identified and characterized as an androgen or glucocorticoid receptor coactivator (ARA55) involved in transcriptional regulation and senescence.16–19 Hic-5 contains four LD (leucine-aspartate repeat) motifs at the amino terminus and four LIM domains at the carboxyl terminus and serves as a multiprotein interacting adaptor protein.11,12 The involvement of Hic-5 in both cytoskeletal organization and transcriptional regulation occurs primarily via the scaffolding properties of this protein.12,15,20,21 Hic-5 exhibits a significant homology to paxillin and binds to FA kinase, protein tyrosine kinase (PYK2), protein tyrosine phosphatase (PTP-PEST), and vinculin, and competes with paxillin to regulate integrin-mediated cell adhesion, actin cytoskeletal organization, contractile activity, and cell spreading.12,20,22–31 Moreover, Hic-5 has been considered as a therapeutic target since elevated levels of this protein have been reported in various diseases,32 and because Hic-5 induces cell plasticity, epithelial to mesenchymal transition (EMT) and fibrosis via Rho/Rho kinase, serum response factor/myocardin transcriptional regulators, and SMAD signaling.14,23 Considering the multiple characteristics of Hic-5 that appear to be relevant to TM cytoskeletal biology, TGF-β signaling, myocilin, Rho GTPase signaling, AH outflow, and steroid-induced glaucoma, we asked ourselves whether Hic-5 plays a critical role in regulation of AH outflow and IOP. To address this possibility, in this initial study we examined Hic-5 expression and distribution in the AH outflow pathway and its regulation and effects on actin cytoskeletal organization, FAs, integrins, myocilin, and fibrogenic activity in human TM (HTM) cells. To the best of our knowledge, this is the first study on Hic-5 demonstrating its regulation and effects in TM cells.

PURPOSE Glucocorticoid (GC)-induced glaucoma is an undesirable side effect of traditional GCs. Ocular hypertension responsible for GC-induced glaucoma is due to alterations in conventional outflow homeostasis. The present study evaluates a novel selective GC receptor agonist (SEGRA), GW870086X, in two different in vitro models of the human conventional outflow pathway. METHODS Primary cultures of human trabecular meshwork (TM) cell monolayers were treated with dexamethasone (DEX), prednisolone (PRED), or GW870086X for 5 days and then assayed for cellular expression and secretion of fibronectin, myocilin, tissue plasminogen activator (tPA), and/or matrix metalloproteinase-2 (MMP2). In parallel, TM cell monolayers on permeable filters treated for 5 days with GCs were assayed for changes in hydraulic conductivity. RESULTS All three GCs increased fibronectin and myocilin secretion in a concentration-dependent manner (P < 0.05). In addition, DEX increased cellular fibronectin and both DEX and PRED significantly increased cellular myocilin (P < 0.0001), while GW870086X did neither. Interestingly, DEX and PRED significantly decreased tPA expression (P ≤ 0.01), while GW870086X had the opposite effect and increased tPA expression in a concentration-dependent manner (P = 0.01). For MMP2, only DEX treatment consistently decreased secretion (P < 0.01). In a functional assay, only PRED treatment significantly decreased hydraulic conductivity of TM cell monolayers (P < 0.05). CONCLUSIONS All three GCs induced differential responses from TM cells. While the novel SEGRA GW870086X increases fibronectin and myocilin secretion similar to two traditional GCs, effects on the matrix degradation enzymes MMP2 and tPA differed significantly, suggesting that GW870086X favors matrix turnover. Consequently, effects on conventional outflow homeostasis may also be dissimilar.

Glaucoma is the second leading cause of visual impairments and blindness worldwide, affecting approximately 70 million people, and is the leading cause of blindness among African Americans in the United States.1 Primary open-angle glaucoma (POAG) is the most common form, accounting for approximately 70% of all cases worldwide. It is characterized by progressive loss of retinal ganglion cell (RGC) axons and irreversible loss of vision,2 which is often associated with elevated intraocular pressure (IOP). Although the exact mechanisms that lead to elevated IOP are poorly understood, an increased resistance to outflow of aqueous humor through the drainage structures in the iridocorneal angle (the trabecular meshwork and Schlemm's canal) is thought to be the major cause of IOP elevation in POAG. Mutations in the myocilin gene (MYOC) are the most common known genetic causes of glaucoma.3–5 These mutations, which cause elevated IOP, are responsible for approximately 4% of POAG, and most cases of autosomal dominant juvenile-onset open-angle glaucoma.5,6 Myocilin is expressed in many ocular tissues including the TM cells.5,7–9 The physiological function of myocilin is not completely understood, although recent reports suggested a possible role in cell–matrix interaction.5,9–11 Myocilin knock-out and -in mouse studies have indicated that wild-type myocilin is not necessary for the physiological regulation of IOP and supports the hypothesis that glaucoma associated with the myocilin mutation is the result of an abnormal property of mutant myocilin.7–9,12 In vitro studies suggest that glaucoma-causing myocilin mutants are misfolded and detergent resistant and do not degrade properly.9,13 Several studies have demonstrated that disease-causing myocilin mutants are secretion incompetent,14–16 accumulate in the endoplasmic reticulum (ER) and induce ER stress.13,17–19 However, in vivo mechanistic studies in a myocilin mouse model that faithfully replicates the human disease have not been available. We have recently generated a transgenic mouse model (Tg-MYOCY437H) that expresses human MYOC containing the Y437H mutation under the control of the CMV promoter within relevant eye tissues including TM.19 Of note, adult Tg-MYOCY437H mice have elevated IOP and show functional and structural loss of RGCs as well as optic nerve degeneration closely resembling those seen in POAG patients with myocilin mutations. Furthermore, we have demonstrated that ER stress plays a key role in the development of elevated IOP in Tg-MYOCY437H mice.19 Sodium 4-phenylbutarate (PBA) is a small chemical chaperone approved for clinical use in patients with urea cycle disorders and has been shown to reduce protein mislocalization and ER stress in different diseases.20–29 A well-known example of this mechanism is the restoration of the cell surface expression and function of the mutant cystic fibrosis transmembrane conductance regulator protein (CFTR) by PBA.20,29 Several studies have suggested that myocilin-associated glaucoma is a protein-misfolding disease and that chemical chaperones can be used to correct myocilin misfolding. Consistent with this hypothesis, in another study, we demonstrated that systemic PBA prevents glaucoma in Tg-MYOCY437H mice.19 Since PBA is approved by the U.S. Food and Drug Administration (FDA) for oral use in urea cycle disorders and has a good safety profile,30,31 we sought to evaluate whether a topical form of this drug, administered as an eye drop could be used for treatment of glaucoma. The purpose of the present study was to determine whether topical ocular PBA treatment would reduce glaucomatous damage in Tg-MYOCY437H mice.

Glaucoma is a common cause of blindness worldwide and is associated with irreversible degeneration of the optic nerve head. Although the mechanisms contributing to the onset of most glaucomas are multifactorial, a primary risk factor is increased intraocular pressure (IOP).1 IOP is likely elevated because of an abnormal increase in resistance to aqueous outflow in the human trabecular meshwork (HTM).1–3 The HTM is an intricate, three-dimensional structure comprised of extracellular matrix (ECM) possessing intrinsic complex topography and stiffness that supports the overlying trabecular meshwork cells. Many ECM proteins have been implicated in glaucoma including myocilin, transglutaminase (TGM)-2, fibronectin, and angiopoietin-like factor (ANGPTL)-7.4,5 HTM cells are provided with thousands of biophysical cues through cell-cell interactions, cell-ECM interactions, and fluctuations in IOP. It has been previously demonstrated that nano- to submicron scale topography with features similar to native ECMs and basement membranes dramatically altered HTM cell behavior and expression of the ECM proteins, myocilin and versican.6 Mechanically stretching HTM cells, which occurs with increased IOP, has also been shown to upregulate more than 100 genes including secreted protein, acidic, cysteine rich (SPARC), fibronectin, and myocilin.7,8 In situ, HTM cells are exposed to dynamic, compliant substrates that markedly differ from flat, rigid substrates such as glass or tissue culture polystyrene (TCP) which are typically used in laboratory investigations of HTM cellular behavior. A recent study demonstrated that the HTM elastic modulus, a measure of stiffness, is markedly increased in the glaucomatous HTM.3 Substratum elastic modulus has been shown to modulate a variety of fundamental cell behaviors.3,9–13 Hydrogels mimicking the elastic modulus of normal and glaucomatous HTM have been shown to dramatically alter cytoskeletal structure and dynamics, protein expression patterns, cell stiffness, cell behavior, and the cellular response to therapeutic agents such as latrunculin-B (Lat-B).2,14,15 Lat-B is an actin cytoskeleton disruptor that is currently in human clinical trials as a novel glaucoma treatment directed specifically at the trabecular meshwork. The only clinically validated treatments for glaucoma act by lowering IOP by decreasing aqueous humor (AH) production or by decreasing the resistance to AH outflow. With the exception of Lat-B and Rho-kinase inhibitors, these therapeutic approaches do not target the HTM.16 Lat-B is thought to decrease IOP by decreasing the resistance to AH outflow through the HTM.17–21 Recently, it has been demonstrated that HTM cells adhered to stiffer substrates were significantly more responsive to Lat-B due to the increased number of actin stress fibers suggesting that the effects of Lat-B treatment would be most pronounced in glaucomatous eyes with a stiffer HTM.2 In addition, Lat-B treatment significantly diminished HTM cell migration on stiffer substrates.15 However, the effect of substratum stiffness on the expression of ECM proteins before and after Lat-B exposure has not been reported. The purpose of this investigation was to determine the impact of Lat-B treatment in the context of substratum stiffness on the expression of several ECM proteins, myocilin, SPARC, ANGPTL-7, fibronectin, and TGM-2 that have been implicated in glaucoma.4,22,23 A better understanding of the impact that clinically relevant substratum stiffness has on ECM gene and protein expression will inform the design of improved in vitro testing methodologies especially with regard to identification and development of novel glaucoma therapeutics.

Glaucoma is a leading cause of visual impairment and blindness, with elevated intraocular pressure (IOP) as a major causative risk factor. Glucocorticoid (GC) therapy causes morphologic and biochemical changes in the trabecular meshwork (TM), an ocular tissue involved in regulating IOP, which can lead to the development of glaucoma in susceptible individuals (steroid responders). Steroid responders comprise 40% of the general population and are at higher risk of developing glaucoma. In addition, a majority of glaucoma patients are steroid responders. Differential distribution of various isoforms of GC receptor (GR) may be responsible for this heterogeneity in the steroid response. The alternatively spliced GRβ isoform acts as dominant negative regulator of classical GRα transcriptional activity. mRNA splicing is mediated by spliceosomes, which include serine-arginine rich proteins (SRps). The purpose of this study was to determine whether specific SRps regulate levels of these isoforms and thereby GC response in TM cells.Quantitative RT-PCR, Western blot analysis, and immunocytochemistry were used to determine the differential expression of different SRps (SRp20, 30c, and 40) in human normal and glaucomatous TM cell strains. Bioinformatics was used to find putative binding sites for SRp20 and SRp40 on exon 9 of the GR gene. A peptide modulator of splicing (bombesin) and SRp expression vectors were used to modulate SRp levels and determine their effects on GRα/GRβ ratios as well as dexamethasone (DEX) responsiveness via GRE- luciferase reporter activity, fibronectin, and myocilin induction in TM cells.SRp20, SRp30c, and SRp40 regulate GR splicing and the GC response in TM cells. Modulation of SRp levels altered the GRβ/α ratio that correlated with DEX responsiveness. Bombesin decreased SRp20; increased SRp30c, SRp40 levels, and GRβ/α ratio, and suppressed DEX response in TM cells.Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of the GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the normal and glaucoma populations.

Olfactomedin was described almost 20 years ago as a novel 57-kDa glycoprotein exclusively expressed in the frog olfactory neuroepithelium.1 Subsequent experiments by many laboratories demonstrated that olfactomedin contains a domain in its C terminus that is present in many proteins in species ranging from reef-building coral Acropora millepora (the phylum Cnidaria) to Homo sapiens.2–7 This domain has a length of approximately 250 amino acids and was named the olfactomedin domain. There are at least 13 proteins containing the olfactomedin domain in mammals, and these proteins form a family.4,8,9 This family segregates into seven subfamilies on the basis of domain organization, biochemical properties, and expression patterns.4 The most studied olfactomedin domain–containing protein is myocilin. Mutations in the Myocilin gene are found in more than 10% of juvenile open-angle glaucoma cases and in 35% to 4% of patients with adult-onset primary open-angle glaucoma.10–14 Myocilin proteins form subfamily III on a phylogenetic tree.4,9 Three proteins, olfactomedin 1 (Olfm1), olfactomedin 2 (Olfm2), and olfactomedin 3 (Olfm3), form subfamily I. Olfm1 is also known as noelin in chicken and Xenopus,15,16 pancortin in mice,17 olfactomedin-related glycoprotein in rats,18 and hOlfA in humans.5 Olfm2 is also known as OlfC,5 and Olfm3 is also known as optimedin.19 These proteins are the most conserved among other olfactomedin domain–containing proteins. High conservation of the amino acid sequences implies that Olfm1, -2, and -3 proteins have important functions. Indeed, overexpression of Olfm1 during embryonic development causes an excess of neural crest emigration in chicken,15 leads to an expansion of the neural plate and enlargement of the neural tube and retina in Xenopus,20 increases the thickness of the optic nerve and produces a more extended projection field in the optic tectum in zebrafish.21 Inhibition of olfm1 expression by olfm1-specific morpholino oligonucleotides reduces eye size, inhibits optic nerve extension, and increases the number of apoptotic cells in the retinal ganglion cell and inner nuclear layers.21 At the same time, elimination of the central (M region) part of Olfm1 in mice produces a relatively mild phenotype.22 It has been shown that knockdown of olfm2 protein expression by morpholino oligonucleotides produces a phenotype that is very similar to that described for the olfm1 knockdown in zebrafish.23 Genetic data indicate that mutation of the OLFM2 gene in humans leading to the R144Q substitution in the protein sequence is a possible disease-causing mutation in Japanese patients with open-angle glaucoma.24 The T86M substitution in the OLFM2 protein has been associated with colorectal cancers.25 Overexpression of Olfm3 inhibits neurite outgrowth and induces Ca2+-dependent aggregation of NGF-stimulated PC12 cells.26 It has been suggested that the expression of Olfm3 stimulates the formation of adherens and tight junctions and modulates cytoskeletal organization, cell–cell adhesion, and cell migration in the brain and retina.26 In this study, we investigated the properties of mammalian OLFM2. Olfm2 and -1 showed an overlapping expression pattern in the course of eye development in the rat. OLFM2 interacted with Olfm1 and -3 but not with the more distantly related proteins myocilin and gliomedin. Although the R144Q and T86M substitutions did not inhibit secretion of OLFM2, L420S substitution in the olfactomedin domain of OLFM2, which corresponds to the I477S mutation in human myocilin, inhibited its secretion. Moreover, secretion of wild-type Olfm1 or -3, but not myocilin, was inhibited in the presence of this mutation. We suggest that severe mutations in one of the closely related olfactomedin domain–containing proteins (Olfm1-3) blocks secretion and probably activity of the whole subfamily.

To characterize the MYOC genotype correlation with phenotypes in an isolated Greek population with a high incidence of glaucoma.Five hundred thirty-one villagers were enrolled in the study. Participants underwent a comprehensive ophthalmic examination. All three exons of myocilin were bidirectionally sequenced. Power calculations and measured genotype analysis was conducted using the genetic variance analysis program, SOLAR version 4.2, to account for the relatedness between individuals.The participants, 376 of whom were linked in a single 11-generation pedigree, ranged in age from 10 to 95 years with a mean age of 49. Sixty-five individuals had POAG, and 27 of those carried the Thr377Met MYOC mutation. Both peak intraocular pressure and vertical cup-to dis- ratio were significantly associated with the MYOC Thr377Met variant (P = 9 x 10(-14) and P = 9 x 10(-8), respectively), whereas central corneal thickness showed no significant association (P0.7).This village had a high frequency of glaucoma, with 12% of the participants aged 10 to 95 years having the disease. In this cohort, the Thr377Met MYOC mutation was significantly associated with both high intraocular pressures and high vertical cup-to-disc ratios. No association was found with central corneal thickness.

Myocilin is an extracellular glycoprotein present in muscular and ocular tissues such as iris, ciliary body (CB), and trabecular meshwork (TM).1–6 It forms large molecular aggregates in the aqueous humor (AH).7–9 Mutations in the myocilin (MYOC) gene cause autosomal dominant juvenile glaucoma, and they are also present in a reduced proportion (3%–5%) of adult-onset primary open-angle glaucoma (POAG) in different populations.10–12 POAG is a heterogeneous disease originated by the progressive apoptosis of the optic nerve,13 which constitutes a leading cause of blindness in developed countries. Myocilin presents a modular design composed of the N-terminal region (amino acids 1-202), the central linker domain (amino acids 202-244),14,15 and the compact16 C-terminal olfactomedin-like domain (amino acids 244-504).3 The N-terminal domain contains two coiled-coil (CC) domains17 and a leucine zipper-like motif3 in the second CC. These three modules are encoded by exons 1, 2, and 3 and coincide approximately with three independent folding domains.17 We have reported that recombinant myocilin undergoes an intracellular endoproteolytic cleavage by calpain II in the central linker domain of the polypeptide chain.15 The processing takes place in the endoplasmic reticulum and releases two fragments that contain the N- and C-terminal structural domains. The C-terminal fragment has been detected in both human and bovine CB and the AH, indicating that the proteolytic processing also occurs in ocular tissues.15 Of note, photomedin-1 and gliomedin, two olfactomedin-like domain–containing proteins, are cleaved in a similar fashion.18,19 Pathogenic mutations located in the olfactomedin-like domain reduce the specific cleavage of the recombinant protein.15 Despite many efforts having being made since the discovery of MYOC as a glaucoma gene in 1997,10 the function of this protein in normal and glaucomatous eyes remains poorly understood. Similarly, the functional meaning of the proteolytic processing of myocilin is currently unknown, although it has been suggested to contribute to the modulation of myocilin interactions.15 In the present study, the specific proteolytic cleavage of recombinant myocilin reduced its extracellular covalent aggregates. In addition, the results revealed the existence of noncovalent interactions between myocilin aggregates, which may play an important role in the extracellular function of the protein.

To developed a genetic mouse model of primary open-angle glaucoma induced by expression of mutated human myocilin in transgenic mice and to test whether expression of mutated human myocilin in the eye angle structures produces more significant damage to the eye than does mutated mouse myocilin.Recombineering in Escherichia coli was used to introduce the Tyr437His point mutation into a BAC carrying the full-length human MYOCILIN (MYOC) gene and long flanking regions. This BAC was used to produce transgenic mice. The expression of myocilin in the iridocorneal angle tissues and aqueous humor was studied by immunohistochemistry and Western blot analysis. Intraocular pressure was measured noninvasively with a fiber optic transducer. Retinal ganglion cells were retrograde labeled with fluorescent gold, and counted 5 days after labeling.BAC transgenic mice expressed elevated levels of myocilin in tissues of the iridocorneal angle. Expression of mutated myocilin induced its intracellular accumulation and prevented secretion of both mutated and wild-type myocilin into the aqueous humor. Transgenic mice demonstrated a moderate elevation of intraocular pressure, which was more pronounced at night than in daytime. In the peripheral retina, transgenic mice lost 20% of the retinal ganglion cells and 55% of large retinal ganglion cells. Axonal degeneration was observed at the periphery of the optic nerve.Expression of equivalent levels of mutated human or mouse myocilin in the eyes of transgenic mice produce comparable pathologic changes that are similar to those observed in patients with glaucoma.

The trabecular meshwork of the human eye is believed to be the tissue that regulates intraocular pressure.1 Human trabecular meshwork (HTM) cells line the trabeculae of the inner areas of the outflow pathway and also are present in the cribriform area. The HTM cells can be cultured in vitro for several passages, but some characteristic properties of the cells, such as myocilin mRNA expression, are downregulated when the cells are cultured on normal tissue culture plastic.2 One possibility for this loss of myocilin could be the absence of appropriate topographic cues normally provided to the cells in vivo. The in vivo basement membrane on the trabeculae and the extracellular matrix at the cribriform region are the surfaces with which HTM cells interact. In contrast to flat plastic surfaces, basement membranes have been shown to have a topographically rich composition at the nanoscale level so that when cells interact with their physical environment, they receive thousands of topographic cues from the surfaces on which they grow.3– 6 Immunomicroscopy7 and scanning electron microscopy8 studies suggest that the trabecular meshwork cells interact with features on the nanometer-scale level. The phenotypic and molecular changes in cells grown on nanosurfaces demonstrate that the topographic features on which the cells are grown profoundly influence them.5,9 –12 These topographic cues are missing on the flat tissue culture plastic normally used to culture HTM cells. It is now possible to fabricate surfaces that have features in the nanometer range to study the interaction of biochemical and physical cues that cells may obtain from their surrounding milieu. These surfaces can finally be produced in large enough quantities for a variety of assays that allow identification of molecular and biochemical properties of cells. Currently, several nanoscale patterns for cell growth can be fabricated.13,14 Synthetic extracellular matrix surfaces are commercially available; however, these are not of uniform topography (Donaldson, Minneapolis, MN). One of the patterns that has been studied is an anisotropic array of ridges and grooves.15–18 The distance between the ridges is termed pitch and is composed of the width of one ridge plus one groove (Fig. 1). The depth of the groove has been shown to be important in directing contact guidance for cells. Loesberg et al.19 reported a threshold of 35 nm of depth to be necessary before a cell is influenced by the substrate pattern, whereas our laboratory found threshold depth to be cell-type dependent and to range from 75 to 150 nm.20 In the study reported herein, all the surfaces had depths of 300 nm. A biomimetic surface can have a variety of pitches as well as differences between the size of the grooves and the ridges. The surfaces used for this study, however, used pitches that each had equal dimensions for the grooves and ridges. Figure 1 Schematic diagram of a patterned surface. Pitch is defined as the sum of the distance of one ridge and one groove. The patterned surfaces used in this report had equivalent dimensions for the grooves and ridges. The depth for all the patterned surfaces ... The purpose of this study was to determine whether HTM cells would be influenced by nanoscale topography compared with flat surfaces. The effect of growth conditions was investigated using several parameters. The alignment and elongation of the HTM cells were parameters that could be compared on a variety of different pitches. At the molecular level, the influence of nanoscale cues on myocilin expression was determined, since data suggest this protein is secreted by HTM cells.21,22 In addition, isoforms of the protein versican appear to be altered when cells are grown in vitro23 and have recently been shown to be influenced by mechanical stretching.24 Changes in versican isoforms, which possess variable numbers of chondroitin sulfate– binding domains, have the potential to influence glaucoma, since it has been reported that chondroitin sulfate increases with disease.25 Therefore, the influence of topography on the versican isoforms was also investigated.

Purpose To evaluate the effect of chemical chaperones on the trafficking of secretion-incompetent primary open-angle glaucoma-associated mutant myocilin and the possibility to rescue cells coexpressing mutant and wild-type myocilin from endoplasmic reticulum (ER) stress and apoptosis. Methods CHO-K1, HEK293 and human trabecular meshwork cells were transfected to express wild-type or mutant (C245Y, G364V, P370L, Y437H) myocilin-green fluorescent protein fusion protein and were treated or not with various chemical chaperones (glycerol, dimethylsulfoxide, or sodium 4-phenylbutyrate) for different time periods. The secretion, Triton X-100 solubility, and intracellular distribution of wild-type and mutant myocilin were analyzed by immunoprecipitation, Western blotting, and confocal double immunofluorescence. The effect of sodium 4-phenylbutyrate on ER stress proteins and apoptosis was examined in cells coexpressing mutant and wild-type myocilin. Results Treatment with sodium 4-phenylbutyrate, but not with glycerol or dimethylsulfoxide, reduced the amount of detergent-insoluble myocilin aggregates, diminished myocilin interaction with calreticulin, and restored the secretion of mutant myocilin. Heteromeric complexes formed by mutant and wild-type myocilin induced the ER stress-associated phosphorylated form of ER-localized eukaryotic initiation factor (eIF)-2alpha kinase and the active form of caspase 3, which resulted in an increased rate of apoptosis. Sodium 4-phenylbutyrate treatment of cells coexpressing mutant and wild-type myocilin relieved ER stress and significantly reduced the rate of apoptosis. Conclusions These findings indicate that sodium 4-phenylbutyrate protects cells from the deleterious effects of ER-retained aggregated mutant myocilin. These data point to the possibility of a chemical chaperone treatment for myocilin-caused primary open-angle glaucoma.

Approximately 1 in 30 unselected patients with open-angle glaucoma (OAG) have a mutation in the myocilin gene. The purpose of this study was to describe the morphologic features of the optic nerve head (ONH) in myocilin glaucoma.A case-control design was adopted. Sixty-six patients heterozygous for a range of myocilin mutation (cases) were matched in disease severity to 105 patients with OAG known not to have a myocilin mutation (controls), using visual field findings. Quantifiable analysis of the ONH was undertaken of stereoscopic photographs, by using custom software with a z-screen. Subjective grading of the cup depth, lamina cribrosa pore shape and orientation, and the slope of the neuroretinal rim was performed by an examiner masked to the subject's mutation status. Mutation screening was conducted using either direct sequencing or single-stranded conformation polymorphism analysis.Patients with a myocilin mutation had glaucoma diagnosed earlier (P0.001) and had higher maximum recorded intraocular pressures (P0.001) than did the control OAG subjects. There was no significant (P0.05) difference in global disc area, global neuroretinal rim area, alpha-parapapillary atrophy, beta-parapapillary atrophy, slope of neuroretinal rim, or visible lamina cribrosa morphology between myocilin mutation carriers and patients with nonmyocilin glaucoma. Disc hemorrhages were identified more frequently in those without mutations (14/209 vs. 1/129), though this was not significant after correction for multiple hypothesis testing.No major structural or morphologic difference of the ONH was detected in pooled data from subjects who had myocilin mutations compared with data from individuals with nonmyocilin glaucoma.

Purpose To map the disease-associated locus of a family with autosomal dominant juvenile-onset primary open-angle glaucoma (JOAG). Methods A complete ophthalmic examination was conducted, and genomic DNA was obtained from 25 members of a Chinese family, of which eight were confirmed as having JOAG. Myocilin (MYOC), optineurin (OPTN), and WD repeat-domain 36 (WDR36) were screened for sequence alterations, by PCR and direct sequencing. Subsequently, a genome-wide scan was performed (Prism Linkage Mapping Set MD-10; Applied Biosystems, Inc., Foster City, CA). Two-point and multipoint linkage analyses were performed with the MLINK, ILINK, and LINKMAP programs. For fine mapping, additional markers flanking the most promising region on 15q were analyzed. The significance of the lod score was tested with simulation analyses by using FASTLINK. Haplotypes were constructed with Simwalk2. Three candidate genes, NR2E3, SMAD6, and CLN6, located within the critical region, were screened for mutations. Results MYOC, OPTN, and WDR36 mutations were excluded in all family members. A maximum two-point lod score of 3.31 at theta = 0.0 was obtained for the marker D15S125. Four adjacent markers, rs2030040, rs169169963, D15S153, and D15S131, gave two-point lod scores of 2.41, 2.90, 3.02, and 2.68, respectively, at theta = 0.0. Haplotype analysis and recombination mapping further confined this region to 15q22-q24 within a genetic distance of 16.6 Mb flanked by D15S1036 and rs922693. No mutations were found in the coding exons and splicing junctions of NR2E3, SMAD6, and CLN6. Conclusions The results provide evidence for the mapping of a novel locus for JOAG at 15q22-q24. A further search for the disease-causing gene in this new JOAG locus is in progress.

PURPOSE The present study compared properties of wild-type and mutated mouse and human myocilin (Myoc) proteins as a prerequisite for development of a mouse model of glaucoma. METHODS cDNA encoding full-length mouse Myoc was cloned into the p3XFLAG-CMV-14 vector. Tyr423His and Ile463Ser mutations were introduced into the mouse Myoc protein by in vitro mutagenesis. Intracellular localization and secretion of wild-type and mutated mouse Myoc proteins were studied in immunostaining and Western blotting experiments, respectively, after transfection into COS-7 cells. RESULTS Similar to human MYOC, wild-type and mutated mouse Myoc demonstrated vesicular staining in transfected cells. However, while wild-type human and mouse Myoc were preferentially located in both the endoplasmic reticulum and Golgi, mutated human and mouse Myoc were located mainly in the endoplasmic reticulum and were excluded from Golgi. Similar to mutations in human MYOC, mutations in mouse Myoc dramatically reduced its secretion from transfected cells. Secretion of mutated Myoc was partially restored by culturing cells at 30 degrees C instead of 37 degrees C. The presence of mutated human MYOC prevented secretion of wild-type mouse Myoc but did not dramatically affect secretion of alkaline phosphatase, thrombospondin, Timp3 or olfactomedin-1. CONCLUSIONS Properties of the mouse Myoc protein are similar to those of the human MYOC. The presence of mutated mouse or human Myoc does not block a general secretory pathway. Expression of mutated Myoc in the eye in mice may mimic human glaucoma and lead to development of a genetic mouse model of glaucoma.

Purpose A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. Methods A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. Results Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. Conclusions Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.

Purpose Glucocorticoid administration can lead to increased intraocular pressure in greater than 90% of patients with primary open-angle glaucoma (POAG), compared with 30% to 40% of the general population. The molecular mechanisms for increased steroid responsiveness among patients with glaucoma are unknown. An alternative splicing variant of the human glucocorticoid receptor GRbeta has dominant negative activity and has been implicated in a variety of steroid-resistant diseases. GRbeta also may play a role in glucocorticoid hyperresponsiveness in glaucoma. Methods Western blot analysis was performed to detect the expression of GRalpha and GRbeta in TM cells and its regulation by dexamethasone (DEX). Immunocytochemistry was used to compare the subcellular expression of GRbeta between normal and glaucomatous TM cell lines. DEX transgene induction in a luciferase reporter was performed to investigate the differential glucocorticoid responsiveness between multiple normal and glaucomatous TM cell lines. Overexpression of GRbeta was conducted in glaucomatous TM cell lines, and the regulation of GRbeta in the Dex-induced reporter gene luciferase or endogenous myocilin and fibronectin expression were determined. Results Trabecular meshwork (TM) cell lines derived from normal individuals expressed higher levels of GRbeta than did glaucomatous TM cells. Glaucomatous TM cells were more susceptible to DEX induction of a luciferase reporter gene than were TM cells derived from normal donors. Overexpression of GRbeta in glaucomatous TM cells inhibited DEX induction of a luciferase reporter gene as well as the endogenous genes MYOC and fibronectin. Conclusions The decreased amount of GRbeta in glaucomatous TM cells could result in enhanced glucocorticoid responsiveness and ocular hypertension.

PURPOSE To obtain experimental in vivo information on the functional properties of myocilin for aqueous humor (AH) outflow and to study in vivo the processing of mutated Tyr437His myocilin. Myocilin is a secreted glycoprotein that is mutated in some forms of primary open-angle glaucoma (POAG), and patients with the Tyr437His mutation have severe phenotypes. METHODS The chicken betaB1-crystallin promoter was used to overexpress wild-type human myocilin and mutated Tyr437His myocilin in the lenses of transgenic mice. Expression of transgenic mRNA was monitored by Northern blot analysis and in situ hybridization. The localization and secretion of transgenic myocilin was investigated by Western blot analysis and light and electron microscopy. Intraocular pressure (IOP) was measured by anterior chamber cannulation. RESULTS Two independent lines were established with each of the constructs that showed a strong expression of transgenic mRNA in their lenses. Transgenic expression resulted in a 4.7 +/- 1.8-fold increase of secreted normal myocilin in mouse AH, compared with its concentration in human AH. Immunoreactivity for transgenic myocilin was observed along the surfaces of lens and corneal endothelium, and in the chamber angle. At 12 weeks of age, the ultrastructure of the trabecular meshwork in mice expressing normal myocilin was not different from that of control eyes, and IOP of transgenic animals did not significantly differ from that of control littermates. In contrast, mutated Tyr437His myocilin was not secreted from lens fibers, but accumulated in dilated cisterns of rough endoplasmic reticulum. Although no structural changes were observed in lenses of animals expressing normal myocilin, lenses with Tyr437His expression developed nuclear cataracts, completely lost transparency, and eventually ruptured. Structural changes in lenses of Tyr437His expressing mice were correlated with a significant increase in IOP. CONCLUSIONS The results do not support the concept that increasing amounts of myocilin in the outflow tissues obstruct the system and directly cause an increase in outflow resistance. Mutated Tyr437His myocilin is not secreted in vivo and causes severe alterations of cellular structure and function. A comparable mechanism may cause POAG in patients with myocilin mutations.

To investigate mechanism(s) by which mutations in the olfactomedin domain of myocilin (MYOC), also known as the trabecular meshwork-induced glucocorticoid response (TIGR) gene, cause autosomal dominant open-angle glaucoma, the structure and properties of wild-type (WT) MYOC protein were examined, when expressed alone or simultaneously with the Q368X or K423E disease-associated polypeptides.Myocilin was analyzed in human aqueous humor and human trabecular meshwork (HTM) tissues. COS-7 and immortalized human trabecular meshwork (iHTM) cell lines were transfected with expression vectors encoding WT MYOC, mutated, and/or epitope-tagged cDNAs. MYOC proteins were characterized by double-epitope tagging procedures and/or Western blot analysis.MYOC polypeptides formed highly similar oligomers in aqueous humor, HTM tissues, transfected COS-7, and iHTM cell lines. These complexes ranged in size from 116 kDa to more than 200 kDa. The smallest complex, approximately 116 kDa, resulted from dimerization between two MYOC monomers. Expression of a 150-kDa complex was strongest in aqueous humor. Cotransfections of the WT construct with either the Q368X or K423E cDNA produced MYOC(WT)/MYOC(mutant) heterodimers and higher molecular weight hetero-oligomeric complexes. WT homo-oligomeric complexes were secreted in the extracellular media of both cell lines whereas the Q368X and K423E mutant/mutant homomultimers and heteromeric WT/mutant oligomers remained sequestered intracellularly.Formation of heteromeric WT/mutant complexes may provide a critical mechanism by which mutant myocilin polypeptides produce autosomal dominant open-angle glaucoma. The intracellular sequestration of abnormal WT/mutant complexes could lead to the malfunction of MYOC-expressing cells and to POAG potentially involving a dominant negative effect.

PURPOSE. To characterize gene expression pattern in the combined tissues of the rat iridocorneal angle by expressed sequence tag (EST) analysis, as part of the NEIBank project. METHODS. RNA was extracted from dissected tissues of the rat iridocorneal angle (iris, ciliary body, trabecular meshwork, and Schlemm’s canal) and used to construct unamplified, nonnormalized cDNA libraries in the pSPORT1 vector. Approximately 5000 clones were sequenced from the 5-end. Clones were clustered and identified using the GRIST software, a procedure based on BLAST comparisons. Complete sequences of several novel cDNAs showing eye-preferred expression patterns were obtained. The expression patterns of several genes have been investigated by Northern blot and in situ hybridization, as well as by RT-PCR. RESULTS. After analysis and removal of non-mRNA sequences, 2195 independent clusters, potentially representing individual eye angle-expressed clones were obtained. The expression profile of the combined rat eye angle tissues was more similar to that of the human iris than to human trabecular meshwork. Several cDNAs encoding transcription factors essential for normal eye development and function including Pax-6, Six3, c-Maf, Maf1, Sox-4, Foxc1, Rx, and Ldb2 were present among sequenced clones. A number of tested cDNAs showed eyepreferred expression patterns. Myocilin, which is abundant in human eye angle tissues, was not observed in the rat collection; however, transcripts for three other olfactomedin-domain proteins were seen. Latrotoxin receptor (CL1AA) and optimedin were shown to be expressed in the iris and ciliary body, as well as in the ganglion and inner nuclear cell layers of the retina, whereas the rat orthologue of the human HNOEL-iso gene was expressed in the iris and sclera and less actively in the trabecular meshwork, retina, and optic nerve. CONCLUSIONS. The iridocorneal libraries are a good source of novel uncharacterized genes and molecular markers for the tissues of the eye angle. Although myocilin is not abundantly expressed in rat eye angle, other olfactomedin-containing genes are expressed there and may play important roles in normal eye function and disease. (Invest Ophthalmol Vis Sci. 2004;45:3081–3090) DOI:10.1167/iovs.04-0302

PURPOSE. To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS. Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O2) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS. The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS. The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma. (Invest Ophthalmol Vis Sci. 2004;45:2652‐2659) DOI:10.1167/iovs.03-0572

Purpose To examine ultrastructurally the composition of major extracellular matrix (ECM) components and the distribution of myocilin in the trabecular lamellae of corneoscleral (CS) meshwork in normal human eyes. The codistribution of myocilin with ECM components was also investigated. Methods Postembedding immunoelectron microscopic studies were performed with antibodies against myocilin and other ECM components, including fibronectin, laminin, vitronectin, tenascin, elastin, fibrillin-1, microfibril-associated glycoprotein (MAGP)-1, decorin, versican, hyaluronic acid, and five types of collagen (I, III, IV, V, and VI). Double labeling of myocilin with other ECM components was performed with different sized gold particles. Results In the trabecular beams of CS meshwork, fibronectin, laminin, and collagen type IV were associated with basement membranes, whereas elastin was specifically localized to the core of elastic-like fibers. Several types of collagens, glycoproteins, proteoglycans, and hyaluronic acid were detected both in the collagen fibers and ground substances. Myocilin predominantly localized in the long-spacing collagens and sheath materials surrounding elastic-like fibers, codistributed with fibronectin, fibrillin-1, MAGP-1, decorin, and type VI collagen. Conclusions This study illustrated the composition of ECM materials in the trabecular lamellae of CS meshwork. Myocilin was specifically localized to long-spacing collagens and the surrounding sheath of elastic-like fibers interacting with microfibril-associated elements where changes have been documented to occur in glaucomatous and aging eyes.

Purpose Mutations in the MYOC gene coding for myocilin are associated with elevated intraocular pressure (IOP), and recombinant myocilin, produced in a prokaryotic expression system, has been reported to affect aqueous outflow facility. This study was conducted to test whether perfusion with a fragment of recombinant myocilin (containing the full-length olfactomedin domain), produced in a eukaryotic expression system, affects facility. Methods 293 EBNA cells were transfected by a vector containing the BM40 signal peptide, a human cDNA coding for myocilin, and a polyhistidine tag (HisTag) sequence. Recombinant protein was isolated by Ni-chelate chromatography, and characterized, and perfused into cultured anterior segments of human and porcine eyes. Results Recombinant myocilin was secreted as a approximately 55-kDa intact protein and two fragments arising from cleavage of the recombinant protein at amino acid 215. The C-terminal fragment, containing the entire olfactomedin domain, was successfully isolated. When perfused into human and porcine eyes, this C-terminal fragment did not appreciably affect outflow facility. Conclusions Although the olfactomedin domain appears to be important for the function of myocilin, perfusion with a recombinant myocilin fragment containing this domain does not change outflow facility. It is possible that both the olfactomedin and N-terminal domains (including the leucine zipper) must be present for myocilin to have full function. Alternatively, posttranslational modifications of myocilin may have a major impact on protein function.

PURPOSE To identify myocilin (TIGR/MYOC) properties that are specific to the human trabecular meshwork (HTM). To search for genes highly expressed in dexamethasone (DEX)-induced HTM cells that are barely expressed or absent in DEX-induced cells from other tissues. METHODS TIGR/MYOC induction by DEX (10(-7) M for 8-10 days) was analyzed by Northern and Western blot analyses in HTM, human umbilical vein endothelial cells, HeLa cells, and human embryonic skeletal muscle cells and optic nerve head (ONH) astrocytes at confluence. Processing and secretion were analyzed after the cells were infected with adenoviruses overexpressing wild-type and mutant forms of TIGR/MYOC. Affymetrix U95Av2 GeneChips (n = 6) and software were used to compare paired expression profiles of HTM, HTM-DEX, ONH astrocytes, and ONH astrocytes-DEX. Identification of HTM-DEX-specific genes (compared with ONH astrocytes-DEX) was performed by selecting genes with the highest fold change values (>/=20). Genes with fold change values of four or more were matched with loci linked to glaucoma, by using gene databases. RESULTS TIGR/MYOC induction by DEX occurred only in HTM cells. Secretory and glycosylation characteristics remained the same across cell types. Expression profile analysis revealed multiple genes differentially upregulated in HTM-DEX including, in addition to TIGR/MYOC, a serine protease inhibitor (alpha1-antichymotrypsin), a neuroprotective factor (pigment epithelium-derived factor), an antiangiogenesis factor (cornea-derived transcript 6), and a prostaglandin synthase (prostaglandin D(2) synthase). Fifteen of the 249 genes with fold change values of four or more mapped to glaucoma-linked loci. CONCLUSIONS The induction of TIGR/MYOC by DEX is HTM-specific, whereas its secretory and glycosylation characteristics are ubiquitous. The known functions of HTM-DEX-specific genes reveal the presence of protective and damaging mechanisms for regulation of IOP during DEX treatment. Besides TIGR/MYOC, other HTM-DEX-specific genes may be good candidates for linkage to glaucoma.